This project is based on the working hypothesis of the program project that the p70S6 kinase and the JARID1B histone demethylase enzymes play key roles in both melanoma cell growth and survival. The studies proposed here will delineate the biochemical and structural basis for the activities of these enzymes and use this information to develop small molecule inhibitors to study the function of these proteins in vivo using melanoma models through the other projects of the program. These studies will also provide lead compounds for development for therapy of melanoma. Aim 1 will focus on p70S6K and Aim 2 will focus on JARID1B. For Aim 1, we have prepared active recombinant full-length p70S6 kinase as well as several truncated protein constructs, prepared a lead p70S6K organometallic inhibitor scaffold (compound EMS) with promising selectivity profile and an IC{50} value in the mid-nanomolar range, and determined the crystal structure of the p70S6 kinase domain bound to the EMS inhibitor. We propose to: (a) biochemically characterize the catalytic activity of the p70S6 kinase; (b) prepare a focused library around the EMS organometallic p70S6 kinase inhibitor for screening in vitro and in vivo (together with Project 2) and determine the X-ray crystal structure of p70S6K/inhibitor complexes; (c) carry out a high-throughput solution screen for organic p70S6 kinase inhibitors, establish the mode of inhibition, and determine the X-ray crystal structure of the p70S6 kinase domain bound to lead organic inhibitors; and (d) use structure-based design, medicinal chemistry, kinase profiling and cell-based studies (together with Project 2) to develop potent and selective second-generation organometallic and organic p70S6 kinase inhibitors. For Aim 2, we have prepared recombinant and enzymatically active JARID1B and the yeast Jhd2p ortholog for biochemical and crystallographic analysis and have prepared preliminary JARID1B and Jhd2p crystals. We now propose to (a) biochemically characterize the catalytic activity of JARID1B and Jhd2p; (b) determine the X-ray crystal structures of JARID1B and Jhd2p and carry out structure-based mutagenesis to facilitate a structure-function analysis; (c) use the recombinant JARID1 proteins (JARID1B and Jhd2p) and structures to facilitate high throughput solution and in silico small molecule inhibitor screening, respectively, and JARID1 inhibitors will be tested in cells together with Project 1 of the program; and (d) crystallize the most potent JARID1 inhibitors with the JARID1 proteins for structure-based inhibitor optimization. Together, the studies proposed here will provide novel molecular insights and small molecule reagents to delineate the roles of two novel protein targets, p70S6K and JARID1B, in melanoma and will provide lead small molecule compounds to develop therapeutic agents to treat melanoma.